1. Volume 46, Issue 2, Pages 226-237 (April 2012)
Genome-wide Functional Annotation of Dual-Specificity Protein- and Lipid-Binding Modules that Regulate Protein Interactions 
Yong Chen, Ren Sheng, Morten Källberg, Antonina Silkov, Moe P. Tun, Nitin Bhardwaj, Svetlana Kurilova, Randy A. Hall, Barry Honig, Hui Lu, Wonhwa Cho 
Molecular Cell 
Volume 46, Issue 2, Pages (April 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 46, 226-237DOI: (10.1016/j.molcel.2012.02.012)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1 Quantification of Residue-Specific Features
(A) Residue and cumulative score obtained from the RFC matrix for the third PDZ domain of PSD95.
(B) The structure of the domain along with electrostatic isosurfaces. Key membrane-binding residues identified by the scoring system are highlighted and labeled.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Membrane-Binding Statistics for 2,000 PDZ Domains Found in 20 Species
Predicted number of membrane-binding PDZ domains is shown for each species. SVM classifier was used for prediction with all features included and Kd = 1 μM as a threshold.
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3 Functional Classification of Membrane-Binding PDZ Domains
(A) The energy-minimized model structure of the SAP102-PDZ3-peptide(RTTPV)-Ins(1,4,5)P3 ternary complex. Electrostatic surface potential (left) and the ribbon diagram (right) show the separation of the peptide- and lipid-binding sites. In the ribbon diagram, R449, R459, and R484 that form the cationic groove are shown in space-filling representation and labeled.
(B) The energy-minimized model structure of the rhophilin 2-PDZ-peptide(EYLGLDVPV)-Ins(1,4,5)P3 ternary complex. Electrostatic surface potential (left) and the ribbon diagram (right) show the proximity of the peptide- and lipid-binding sites. In the ribbon diagram, K576 and K579 in the α2A helix that are involved in lipid binding are shown in space-filling representation and labeled.
(C) The energy-minimized model structure of tamalin-PDZ-peptide(IRDYTQSSSSL) binary complex. Because of severe steric clash, an Ins(1,4,5)P3 molecule could not be docked on the PDZ-peptide complex. In the ribbon diagram, R166, H167, and R168 in the α2A helix that constitute the lipid-binding site are shown in space-filling representation and labeled. Electrostatic calculations were performed using GRASP2 (Petrey and Honig, 2003). Blue and red colors indicate positive and negative electrostatic potential, respectively. See the Supplemental Experimental Procedures for molecular docking procedures.
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6. Figure 4
Effects of Lipid Binding of Each Class of the PDZ Domain on Its Peptide Binding
(A) Binding of class A SAP102-PDZ3 to F-Ahx-RTTPV in the absence (filled symbols) and presence (open symbols) of 150 μM PM-mimetic vesicles.
(B) Binding of class B1 rhophilin 2-PDZ to F-Ahx-EYLGLDVPV in the absence (filled symbols) and presence (open symbols) of 150 μM PM-mimetic vesicles.
(C) Binding of class B2 tamalin-PDZ to F-Ahx-IRDYTQSSSSL in the absence (filled symbols) and presence (open symbols) of 150 μM PM-mimetic vesicles. The peptide concentration was 5 nM. Notice that for the class A and B1 PDZ domains, vesicles have a modest to no effect on peptide binding, whereas for the class B2 PDZ domain, vesicles greatly interfere with the peptide binding. See Table S4 for Kd values and the Experimental Procedures for experimental details.
Molecular Cell  , DOI: ( /j.molcel )
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7. Figure 5
Effects of Lipid Binding of Rhophilin 2-PDZ on the Cellular Localization and Function of Rhophilin 2
(A) Membrane binding of rhophilin 2-PDZ WT and mutants. Binding of PDZ domains to PM-mimetic vesicles was measured by SPR analysis. The two mutants show significantly reduced membrane affinity.
(B) GST pull-down assay for rhophilin 2-PDZ WT and K576E/K579E. GST or GST-EYLGLDVPV (5 μg) was incubated with 0–5 μg of rhophilin 2 proteins, and the GST-bound proteins were analyzed by SDS-PAGE and immunoblotting by a rhophilin 2 antibody.
(C) Confocal microscopic images of F-actin (green) and rhophilin 2 (red) in fixed HeLa cells transiently transfected with mRFP-tagged rhophilin 2 WT. Notice that only the rhophilin 2-expressing cell shows distinct morphology and dramatically reduced F-actin stress fibers.
(D and E) Confocal images for K576A/K579A and K576E/K579E mutants, respectively. These mutants show distinctly different subcellular localization patterns from WT and have little effect on F-actin.
(F) The control images taken using HeLa cells transfected with the empty expression vector. White bars indicate 10 μm.
Molecular Cell  , DOI: ( /j.molcel )
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